IN VIVO PHOSPHORYLATION OF BACTERIAL–TYPE PHOSPHOENOLPYRUVATE CARBOXYLASE FROM DEVELOPING CASTOR OIL SEEDS AT THREONINE-4 AND SERINE-451
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Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branchpoint of plant C-metabolism. Plant genomes encode several closely related plant-type PEPC (PTPC) isozymes, and a distantly related bacterial-type PEPC (BTPC). Two physically and kinetically distinct oligomeric classes of PEPC occur in the endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of 107-kDa PTPC subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between Class-1 PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. BTPC functions as a catalytic and regulatory subunit of the allosterically-desensitized Class-2 PEPC, hypothesized to support PEP-flux to malate for leucoplast fatty acid synthesis. Previous studies established that BTPC: (i) subunits of COS Class-2 PEPC are phosphorylated at multiple sites in vivo and (ii) phosphorylation at Ser425 provides a new tier of enzyme control in developing COS. LC MS/MS and LTQ-FT MS identified Thr4 and Ser451 as additional in vivo phosphorylation sites of immunopurified COS BTPC (corresponding to acidophilic and basophilic protein kinase consensus sequences, respectively). Immunoblots probed with a phosphorylation-site specific antibody raised against a synthetic phosphopeptide indicated that Ser451 phosphorylation is promoted during seed development, becoming maximal in stage VII (full cotyledon) COS. Although several pThr4 containing BTPC peptides were non-immunogenic, the collective results indicate that Thr4 is also phosphorylated in vivo. Kinetic effects of each phosphorylation site were examined using phospho-mimetic mutants of heterologously expressed COS BTPC. BTPC’s phosphorylation at Ser451 appears to be inhibitory, as reflected by significantly increased Km(PEP) values, and reduced I50(malate) and I50(Asp) values of a S451D mutant. By contrast, kinetic characterization of a T4D phosphomimetic mutant indicated that Thr4 phosphorylation is not regulatory in nature. However, Thr4 exists in a conserved forkhead-associated (FHA) binding domain (pTXXD) that has received considerable prominence as a phospho-Thr dependent protein interaction module. These results further our understanding of multisite phosphorylation of BTPC in developing COS and its possible contribution to the control of Class-2 PEPC activity.